1. Field of the Invention
The present invention relates to purified and isolated citrus blight leaf proteins useful for the detection of citrus blight. In particular, the present invention relates to the purified and isolated citrus blight leaf proteins having a partially known amino acid sequence, antigens prepared using the citrus blight leaf proteins, antibodies prepared using the antigens and methods of diagnosis of citrus blight using electrophoresis and immunological techniques.
2. Description of the Art
Citrus blight is a citrus disorder of unknown etiology which causes significant citrus fruit and tree lose. The disease results in overall decline of plant vigor, ultimately leading to the need to remove affected trees and resulting in enormous subsequent economic loss.
Citrus blight is an extensive world-wide problem affecting numerous countries having large citrus industries, especially countries such as Argentina, Brazil, and South Africa, where citrus crops are an important part of the countries' economy. Blight has been known to exist in Florida for well over a century, where despite extensive research on the disease, investigators have failed to identify any causal agent. Citrus blight has been the most important cause of tree loss in Florida citrus orchards in recent years. The estimated annual loss to Florida citrus growers, including tree value and replacement cost, is about $52 million (Timmers, L., First Intl. Seminar on Citrus Rootstocks, San Pablo, Brazil, 1990).
The citrus blight syndrome begins as a delay or lack of new growth flush during the spring and a wilting of existing leaves. The wilting can not be corrected by irrigation. Subsequent seasonal growth exhibits increased leaf water potential, increased abscission of leaves, and death of terminal growing points. The metabolism of the tree is disrupted. Zinc, chlorides and sodium accumulate in the leaves. The enzymes, ribulose 1,5-biphosphate carboxylase and carbonic anhydrase, are also less active in blight stressed trees.
Recent studies have shown that the disorder might be transmitted by root grafting and therefor and infectious agent. In a study where seedlings were infected with Xylella fastidiosa, a xylem-limited, gram-negative bacterium, the seedlings developed some symptoms of blight. However, immature seedlings (&lt;5 years) in the field are not susceptible to infection with the bacterium. Further studies failed to infect reconstituted trees, and citrus blight symptoms did not develop in trees grafted with buds, shoots, or roots from bacteria blighted trees.
In spite of the fact that citrus blight has no clear diagnostic visible symptoms, it has certain determinable characteristics which are used to distinguish it from other tree declines.
The leaves of affected trees exhibit zinc deficiency and zinc accumulates in other tissue, especially the trunks. The method of Wutscher, H. et al Proc. Fla. State Hort. Soc. 90:81-84 (1977) can be used to determine the zinc accumulation in trunk wood as a diagnostic for the disease. The method uses wood rather than bark since the percentage of increase in zinc concentration is much greater than with bark. However, since soils and applications of zinc as a fertilizer vary among orchards, it is essential to compare zinc levels in wood declining tree with those in healthy trees in the same orchard.
Absorption of water by the trunks of blighted trees was found to be abnormal and can be used in the diagnosis of the disease (See Cohen, M et al Proc. Intl. Soc. Citriculture 3:884-886 (1974)). Water flow in most limbs and small branches is comparable to that in healthy trees. However, water flow is impeded in the trunk and large roots and branches except for about the outer centimeter of xylem tissue. Smaller roots may be completely blocked or may show normal water flow. The water flow test for blight was refined and simplified by using a syringe to inject water into a small hole bored into the tree trunk (Lee, R. et al, Plant Dis. 68:511-513 (1984)). Healthy trees and those affected absorb from about 0.25 to 1.0 ml/sec whereas blighted trees absorb virtually no water.
Cytological research has revealed the presence of dark fibrous occlusions and amber, amorphous plugs in the vessels of blighted citrus trees. Amorphous plugs occurred more commonly and appeared to be more characteristic of blighted citrus trees. Brlansky, R. et al Phytopathology 74:1324-1328 (1974) demonstrated that restriction of water flow in blighted trees is most closely correlated to the numbers of amorphous plugs in the vessels and appears to be characteristic of blighted trees. However, caution must be used when identifying amorphous plugs because gum plugs formed in response to injuries or infections can appear very similar to amorphous plugs.
Imposition of stress on plants results in metabolic changes which can alter the types of proteins formed by cells. New proteins can also be induced in plants by the presence of pathogens. Derrick, K. et al (Plant Dis. 74:168-170 (1990)) looked for but did not find blight-specific proteins in either the leaves or bark of blight diseased trees. Derrick et al. did however report the presence in roots of a number of proteins that appear to be unique to blight. Derrick et al. failed to detect the blight-specific proteins in some stem samples and in root samples from two of 17 trees with symptoms. Derrick et al. inferred from this that the blight pathogen is unevenly distributed in infected trees.
The discovery and development of a sensitive and specific biological marker would allow diagnosticians and researchers to differentiate citrus blight from other physiological or pathological disorders and to detect the disease in asymptomatic trees. The ability to accurately diagnose citrus blight is crucial to preventing the enormous economic loss each year attributable to citrus blight. The present techniques for detecting citrus blight require determination of high zinc levels in bark or wood samples, impairment of water movement, and water deficits in leaves. These diagnostic techniques are destructive to young citrus trees due to necessity of repeated sampling and the increased incidence of pathogenic infection at the sampling sites. Furthermore, the current diagnostic techniques are expensive, time consuming and indirect techniques initiated after the blight has expressed itself in recognizable symptoms and well after the blight has started to seriously effect the tree's metabolism.